Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Hematopoiesis, Biological Processes
We recently published a FISH-Flow protocol in which pools of RNA fluorescent in-situ hybridization (FISH) probes are hybridized to nascent (47S) or mature (18S, 28S) rRNA, followed by quantification using flow cytometry (Antony et al, Molecular Cell 2022, Antony et al, STAR Protocols 2023). Using DAPI staining for gating, this protocol allows us to quantify rRNA levels in different phases of the cell cycle. We have now optimized this protocol to be compatible with surface antibody staining and have applied it to mouse bone marrow. To compare rRNA levels between cell types and samples, all quantifications are normalized to the median across total bone marrow (i.e.: median of total marrow = 1 unit). In doing so, FISH-Flow provides precise quantification of nascent and mature rRNA levels in all cell types, with high levels of fidelity across biological replicates.
We identified a 7-fold range of nascent rRNA transcription and a 9-fold range of mature rRNA abundance across normal mouse hematopoietic cell types. This variation reveals a striking pattern between rRNA level and differentiation state. HSCs, despite being quiescent, have nascent rRNA levels at 1.3 units - above the median for total bone marrow - indicating that they are actively transcribing rRNA despite not actively cycling. Nascent and mature rRNA levels increase as HSCs differentiate into MPPs, and progenitor populations such as GMPs and MEPs display the highest nascent and mature rRNA levels (GMP 47S levels at 2.5 units and 18S levels at 3.2 units, MEP 47S levels at 2.2 units and 18S levels at 3.1 units). Terminal maturation of different lineages shows varied dynamics of changes in rRNA levels that plateau at different levels. Our analysis recapitulates the previously reported shutdown of rRNA transcription in terminal erythropoiesis, with orthochromatic erythroblasts exhibiting the lowest rRNA levels (47S and 18S levels at 0.37 units). Differentiated myeloid cells such as neutrophils have nascent rRNA levels of 0.95 units and mature rRNA levels of 0.71 units, and monocytes have nascent rRNA levels of 1.7 units and mature rRNA levels of 2.4 units. Importantly, these differences are not explained merely by cell cycle phase. rRNA differences persist when comparing matched cell cycle subpopulations within cell types, and multiple cell types with similar proliferation rates display substantially different levels of rRNA transcription (e.g. proerythroblasts and polychromatic erythroblasts have 55% of cells in S/G2/M phases but have 2.8-fold differences in 47S rRNA levels). Further, comparison of nascent 47S and mature 18S or 28S levels across cell types reveals that the correspondence across different cell types is not linear, pointing to additional levels of regulation such as mature rRNA stability (ribosome subunit half-life).
Patterns of rRNA abundance are thus a cell-type specific property and are not solely a consequence of proliferation kinetics. In conjunction with previous work from our lab indicating that multiple cell-type-specific transcription factors (CEBPA, PU.1, others) bind and potentially regulate rRNA transcription, these new data are the most detailed quantification of rRNA in hematopoiesis assembled thus far and represent a baseline for future comparisons of how rRNA transcription is regulated in stress and malignant hematopoiesis.
Disclosures: No relevant conflicts of interest to declare.